Drug resistance remains a pressing concern for antimalarial chemotherapies. Management of resistance has largely been a reactive process, but here we propose a proactive approach that exploits predicted evolutionary trajectories to maintain a drug-sensitive parasite population. Our strategy is to design new drugs such that the fitness cost of resistance restricts the ability of mutant parasites to survive combination therapy or to persist in the absence of selection. Through whole organism high-throughput screening we have discovered many new chemotypes that act on mitochondrial targets and either in combination or in a single molecule target both wild-type and mutant parasites and suppress the emergence of resistance in vitro. Here we focus on the Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) target and fully explore the structural, functional, and fitness consequences of drug resistance pathways. We extend the findings from our current award to fully explore mutually incompatible resistance, establishing an in vivo model system to understand the impact of the host environment on the parasite's ability to escape drug pressure. PfDHODH is clinically relevant and has multiple structural classes of inhibitors providing a substantial toolset with which to study our combination strategy in depth. We will identify chemical inhibitors that target wild-type and mutant forms of DHODH and optimize efficacy for in vivo studies. Using an approach that has proven highly productive, we will prioritize the molecules based on cross-resistance screening against a panel of resistant mutants and focus on compounds demonstrating validated potency against both the wild-type and mutants. We will validate the resistance mutations and assess the structural and biological consequences of these mutations by determining the structures of the most prevalent mutant forms, measuring the effects of the mutations on the enzyme, on mitochondrial function, and on the growth of the organism. We will develop a protocol for using a humanized mouse model of P. falciparum infection to measure the most relevant in vivo resistance pathways and test the suppression of drug resistance. This has implications not just for this work, but also for others in the field who are developing methods to prevent the emergence of drug resistance.